Rudd and Hopwood have isolated a series of mutants from Streptomyces coelicolor A3(2) that are blocked in the biosynthesis of the antibiotic actinorhodin (act) or that of a mycelial red pigment of unknown structure (red). A few mutants were also isolated which accumulate a yellow-orange pigment and produce less actinorhodin and red pigment (ora). The act and red mutants were ordered into several classes, many of which accumulate biosynthetic intermediates. Mutants of each class were mapped to the chromosome. Under the proposed project we will isolate the red and the yellow-orange pigment and determine their chemical structures. Guided by a cosynthesis bioassay we will isolate the accumulated biosynthetic intermediates from act and red mutants and determine their structures. The conversion of these intermediates into the endproducts will then be verified by tracer experiments, and the enzymes catalyzing individual conversion steps will be assayed for in wild-type cell-free extracts. Following detection of specific enzymes in the wild-type we will check for their presence in competent strains and their absence in the appropriate blocked mutants. Further analysis will reveal the biochemical nature of the lesion in the various mutants, e.g., absence of enzyme protein, presence of inactive protein.